We will develop competitive antagonists of NPY as pharmacological tools to be used in the understanding of its physiological role. Rat/human NPY is a 36-residue, C-terminally amidated peptide. Other members of the NPY family are peptide YY (PYY) and pancreatic polypeptide (PP) with 64% and 53% sequence homology to r/hNPY, resp. Recent studies by us and others suggest that NPY's actions are mediated through several receptor subtypes which include the Y1 and Y2 receptors. In brief, the concept of multiple receptor subtypes (pre- and post-synaptic) was first proposed by Wahlestedt et al. (1986) who compared the relative potency of NPY, PYY and PYY(13-36) in the guinea-pig iliac vein and rat vas deferens assays. In the rat vas deferens assay, NPY, PYY and PYY(13-36) have approximately the same potency while in the guinea pig iliac vein assay the C-terminal fragment PYY(13-36) is inactive. At the writing of this grant, it is believed that the Y1 receptor as well as a yet not fully characterized receptor on mast cells are responsible for some of the cardiovascular effects observed in vivo after administration of NPY. Further SAR studies suggested that the Y1 receptor needs the structural integrity of both C- and N- termini for high affinity, while PYY13-36 and other smaller C-termini fragments have been identified to bind with high affinity to the Y2 receptor. Established human neuroblastoma cell lines (SK-N-MC and SK-N-BE2) bearing predominantly Y1 and Y2 receptor subtypes resp. will be used to determine binding affinity. SK-N-MC cell lines responding to norepinephrine by secreting cAMP which is inhibited by addition of NPY or its fragments will be used as a functional assay. In order to generate and identify competitive antagonists of NPY, we propose to pursue several specific aims that take advantage of the present understanding of peptide modifications (deletions, substitutions, amide bond surrogates, and structural constraints) that have lead in other peptide families to competitive antagonists. In summary, we will identify the smallest and most constrained peptidic structure that retains full NPY Y2 and Y1 receptor binding affinity. We will alter these structures, such as by introducing amide bond surrogates at those positions identified by an alanine scan to be most sensitive to alteration, in an effort to generate an antagonist . We will then improve the binding affinity of our lead compounds using conventional SAR approaches. This can be achieved through optimization of the ionic, steric, hydro-philic/-phobic character of particular elements of the structure, and by the introduction of additional constraints in the form of backbone and beta-methylated amino acids. We will express the NPY receptor recently characterized (Duman et al.) in order to carry out binding and structural studies. Finally, we will provide selected analogs to investigators for studies of their effects on the control of cardiovascular functions in vivo and in vitro (mast cells and others).